Peptide immobilisation on porous silicon surface for metal ions detection
© Sam et al; licensee Springer. 2011
Received: 9 December 2010
Accepted: 6 June 2011
Published: 6 June 2011
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© Sam et al; licensee Springer. 2011
Received: 9 December 2010
Accepted: 6 June 2011
Published: 6 June 2011
In this work, a Glycyl-Histidyl-Glycyl-Histidine (GlyHisGlyHis) peptide is covalently anchored to the porous silicon PSi surface using a multi-step reaction scheme compatible with the mild conditions required for preserving the probe activity. In a first step, alkene precursors are grafted onto the hydrogenated PSi surface using the hydrosilylation route, allowing for the formation of a carboxyl-terminated monolayer which is activated by reaction with N-hydroxysuccinimide in the presence of a peptide-coupling carbodiimide N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide and subsequently reacted with the amino linker of the peptide to form a covalent amide bond. Infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy are used to investigate the different steps of functionalization.
The property of peptides to form stable complexes with metal ions is exploited to achieve metal-ion recognition by the peptide-modified PSi-based biosensor. An electrochemical study of the GlyHisGlyHis-modified PSi electrode is achieved in the presence of copper ions. The recorded cyclic voltammograms show a quasi-irreversible process corresponding to the Cu(II)/Cu(I) couple. The kinetic factors (the heterogeneous rate constant and the transfer coefficient) and the stability constant of the complex formed on the porous silicon surface are determined. These results demonstrate the potential role of peptides grafted on porous silicon in developing strategies for simple and fast detection of metal ions in solution.
The detection and quantification of heavy metals in the environment are of great importance, due to their high toxicity and their lifetime in soil, air and groundwater. The detection techniques already available are very expensive and difficult to implement. Therefore, there is a real need to develop new detection schemes that are rapid, simple, sensitive and low cost. Electrochemical sensors based on modified surfaces with recognition probes meet these criteria for a fast and easy analysis [1, 2], and they are likely to be miniaturised to allow the development of detection equipment capable of operating directly on site. These devices could then complete or even replace the existing conventional techniques.
Surface modification by immobilisation of organic molecules is a very important step and search of new methods is constantly developing [3, 4]. The molecular structure, the homogeneity of the layer, the surface density, bonds stability and processes reproducibility are parameters that determine the performance of subsequent applications of these modified surfaces and therefore, must be perfectly controlled.
Furthermore, the choice of the appropriate ligands to be immobilised on the electrode surface is a crucial issue. The majority of work in this field involves a tedious synthesis of selective macrocyclic ligands for a target metal . In nature however, metal binding is achieved with high degree of selectivity using peptide motifs .
The known works in this area refer to the immobilisation of peptides on a gold electrode [7, 8]. However, the peptide ligands were self-assembled on the surface via a moderately strong gold-sulphur bond. These monolayers are kinetically labile when exposed to moderate temperatures, a chemical attack or application of a potential . Covalent attachment of monolayers on silicon surface through the formation of silicon-carbon bond is an attractive route, since it offers the best performances in terms of robustness and can be made reproducible, with a high yield . In this framework, the advances performed in silicon surface chemistry allowed for attaching functional groups upon demand [11, 12]. Generally, the surface functionalization requires a multi-step reaction scheme [13, 14]. In addition, the use of porous silicon substrates, allowing for an increased surface interaction area, can enhance the detection signal significantly.
Cyclic voltammetry is an efficient method used extensively to study metal ions complexed to electrodes modified by ligands [15, 16]. Parallel to experimental investigations, theoretical studies have been developed to predict and interpret the electrochemical behaviour of this new type of electrodes [17, 18].
In this work, Glycyl-Histidyl-Glycyl-Histidine (GlyHisGlyHis)-modified PSi was prepared by anchoring the peptide on a carboxyl-terminated PSi surface using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling agents. Electrochemical behaviour of such prepared electrodes was carried out in the presence of copper ions by means of cyclic voltammetry. Electrochemical parameters were determined as well.
Silicon wafers were purchased from Siltronix, Archamps, France. All cleaning and etching reagents were of VLSI grade and supplied by Merck. Other chemicals were purchased from Sigma-Aldrich (Munich, Germany), Acros Organics (Geel, Belgium) or Fluka (Buchs, Switzerland) and were of the highest purity available. The 10 × PBS buffer (pH = 7.4) was obtained from Ambion (Darmstadt, Germany). Ultrapure water (MilliQ Billerica, MA, USA; 18.2 MΩcm) was used for solution preparation and rinses.
The silicon samples of 15 × 15 mm2 size were cut from double-side polished (100) oriented p-type silicon wafers boron doped, 0.08-0.12-Ωcm resistivity and were cleaned in 3:1 96% H2SO4//30% H2O2 (piranha solution) for 15 min at 100°C and copiously rinsed with MilliQ water. The native oxide was removed by immersing the samples in 50% aqueous HF for 1 min. The hydrogen-terminated surfaces were electrochemically etched in a 1/1 50% HF/absolute ethanol mixture for 30 s at a current density of 80 mAcm- 2. The prepared PSi surface was rinsed with MilliQ water and dried under a nitrogen stream.
The freshly prepared PSi sample was transferred into a Schlenk tube containing neat undecylenic acid under argon bubbling and allowed to react at 150°C for 16 h. The PSi surface was subsequently rinsed twice for 30 min in an outgassed Schlenk tube containing acetic acid at 75°C and was blown dry under a nitrogen stream. The surface, now bearing acid terminations, was introduced into a Schlenk tube containing a solution mixture of 5 mM EDC and 5 mM NHS and allowed to react under continuous argon bubbling for 90 min in a water bath at 15°C. The resulting succinimidyl-ester-terminated surface (activated surface) was copiously rinsed with water and dried under a nitrogen stream. The activated surface was immersed in an outgassed Schlenk tube containing a solution of 10- 4 M GlyHisGlyHis peptide in 1 × PBS buffer at pH approximately 7, overnight. The resulting surface was thoroughly rinsed and dried.
The Fourier transform infrared (FT-IR) spectra were recorded using a Bruker (Equinox 55) spectrometer (Ettlingen, Germany) equipped with a deuterated triglycine sulphate detector. The samples were mounted in a purged sample chamber in transmission geometry at normal incidence. All FT-IR spectra were collected with 200 scans in the 900-4,000 cm- 1 spectral region at 4 cm- 1 resolution. Background spectra were obtained by using an untreated deoxidised flat silicon wafer mounted in the same geometry.
The X-ray photoelectron spectroscopy (XPS) spectra were obtained with a Thermo Electron VG ESCALAB 220i XL spectrometer (Thermo Electron Corporation, Waltham, MA, USA), using an Al Kα1 monochromatic X-ray excitation, and providing an overall full width at half-maximum (fwhm) energy resolution of 0.31 eV.
All glassware was rinsed with 6 M HNO3, then thoroughly with MilliQ water to avoid metal ion contamination.
The copper ions were accumulated at the GlyHisGlyHis-modified PSi electrode at open circuit potential by dipping the sample into 10 mL of a stirred aqueous solution of Cu(II) sulphate in acetate buffer (pH = 8) for 15 min. The sample was removed from the solution, thoroughly rinsed with MilliQ water, dried under a nitrogen stream and transferred to the electrochemical cell.
The electrochemical measurements were performed with an Autolab potentiostat using a three-electrode electrochemical cell comprising the modified PSi as working electrode, a platinum wire counter electrode and an Hg/Hg2SO4 reference electrode. The electrolyte was copper-free ammonium acetate at pH 4 adjusted with HCl. The solution was degassed with argon for 15 min prior to data acquisition. Cyclic voltammetry was performed at different sweep rates between -800 and 0 mV.
Cyclic voltammetry is an efficient method to extract kinetic parameters such as heterogeneous rate constants k° and charge transfer coefficients α for surface immobilised redox species by examining the variation of peak potential versus experimental time scale (i.e. scan rate). Data analysis relies on a theoretical methodology developed by Laviron et al. .
Where E° is the standard potential of the surface redox species, v is the scan rate (volt/second), n is the number of transferred electrons, R is the ideal gas constant, T is the temperature and F is the Faraday constant.
These equations have been established by Laviron considering the limiting conditions where the reaction is totally irreversible. He considered that this case corresponds to the experimental condition where δE p > 200 mV , where δE p denotes the peak potential separation. In our case, this condition is fulfilled for scan rates above 0.2 Vs- 1. The data E p,a = f(ln v) of Figure 5a are replotted as Figure 5b by considering the highest scan rates. The plot yields a straight line with a slope equal to RT/(1 - α)nF deduced from Eq. 2 and using the anodic potential peak. The value determined for α is 0.77.
which is valid for E p > 200 mV. The calculated k° value is 1.56 s- 1.
Where I ∞ is the limiting current density corresponding to the saturation of the surface by copper ions, K is the pseudo-adsorption coefficient which represents the apparent stability constant of the complex formed on the PSi surface by binding of copper to the GlyHisGlyHis peptide and C is the Cu2+ concentration in the accumulation solution.
The Langmuir curve (solid line in Figure 6) gives a good fit of the experimental data. The value of the limiting current density obtained is 13.44 μA cm- 2 and the apparent stability constant of the complex Cu-GlyHisGlyHis formed on the PSi surface is K = 3 × 105 M- 1.
The value of I ∞ gives an indication on the sensitivity of the sensor, which has implications for the detection limit whilst the value of K is indicative of the affinity of the peptide for the metal ion and hence determines the usable concentration range of the sensor. As a consequence of the high affinity constant for Cu-GlyHisGlyHis, the final sensor is expected to operate in a low concentration range with a low detection limit.
The GlyHisGlyHis peptide was covalently incorporated onto the PSi structure using multi-step chemistry consisting of: PSi formation, thermal hydrosilylation of undecylenic acid, activation of the acid-terminated surface by formation of a succinimidyl ester, and finally Gly-His-Gly-His anchoring by amidation reaction. Infrared spectroscopy confirmed the efficiency of the process at each stage of surface modification. XPS measurements confirmed the high quality of the grafting and the formation of silicon-carbon covalent bonds. Cyclic voltammetry displayed the ability of the GlyHisGlyHis-modified PSi to complex Cu (II) ions from solution. This result would then demonstrate the role of peptide monolayer in metal detection strategies. The kinetic parameters such as heterogeneous rate constant and transfer coefficient were extracted from the cyclic voltammetry measurements. The apparent stability constant was also determined.
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